The present invention is in the field of secreted proteins that are related to the nodal-related secreted subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Secreted Proteins
Many human proteins serve as pharmaceutically active compounds. Several classes of human proteins that serve as such active compounds include hormones, cytokines, cell growth factors, and cell differentiation factors. Most proteins that can be used as a pharmaceutically active compound fall within the family of secreted proteins. It is, therefore, important in developing new pharmaceutical compounds to identify secreted proteins that can be tested for activity in a variety of animal models. The present invention advances the state of the art by providing many novel human secreted proteins.
Secreted proteins are generally produced within cells at rough endoplasmic reticulum, are then exported to the golgi complex, and then move to secretory vesicles or granules, where they are secreted to the exterior of the cell via exocytosis.
Secreted proteins are particularly useful as diagnostic markers. Many secreted proteins are found, and can easily be measured, in serum. For example, a xe2x80x98signal sequence trapxe2x80x99 technique can often be utilized because many secreted proteins, such as certain secretory breast cancer proteins, contain a molecular signal sequence for cellular export. Additionally, antibodies against particular secreted serum proteins can serve as potential diagnostic agents, such as for diagnosing cancer.
Secreted proteins play a critical role in a wide array of important biological processes in humans and have numerous utilities; several illustrative examples are discussed herein. For example, fibroblast secreted proteins participate in extracellular matrix formation. Extracellular matrix affects growth factor action, cell adhesion, and cell growth. Structural and quantitative characteristics of fibroblast secreted proteins are modified during the course of cellular aging and such aging related modifications may lead to increased inhibition of cell adhesion, inhibited cell stimulation by growth factors, and inhibited cell proliferative ability (Eleftheriou et al., Mutat Res 1991 Mar-Nov;256(2-6): 127-38).
The secreted form of amyloid beta/A4 protein precursor (APP) functions as a growth and/or differentiation factor. The secreted form of APP can stimulate neurite extension of cultured neuroblastoma cells, presumably through binding to a cell surface receptor and thereby triggering intracellular transduction mechanisms. (Roch et al., Ann NY Acad Sci 1993 Sep. 24;695:149-57). Secreted APPs modulate neuronal excitability, counteract effects of glutamate on growth cone behaviors, and increase synaptic complexity. The prominent effects of secreted APPs on synaptogenesis and neuronal survival suggest that secreted APPs play a major role in the process of natural cell death and, furthermore, may play a role in the development of a wide variety of neurological disorders, such as stroke, epilepsy, and Alzheimer""s disease (Mattson et al., Perspect Dev Neurobiol 1998; 5(4):337-52).
Breast cancer cells secrete a 52 K estrogen-regulated protein (see Rochefort et al., Ann N Y Acad Sci 1986;464:190-201). This secreted protein is therefore useful in breast cancer diagnosis.
Two secreted proteins released by platelets, platelet factor 4 (PF4) and beta-thromboglobulin (betaTG), are accurate indicators of platelet involvement in hemostasis and thrombosis and assays that measure these secreted proteins are useful for studying the pathogenesis and course of thromboembolic disorders (Kaplan, Adv Exp Med Biol 1978;102:105-19).
Vascular endothelial growth factor (VEGF) is another example of a naturally secreted protein. VEGF binds to cell-surface heparan sulfates, is generated by hypoxic endothelial cells, reduces apoptosis, and binds to high-affinity receptors that are up-regulated by hypoxia (Asahara et al., Semin Interv Cardiol 1996 Sep; 1 (3):225-32).
Many critical components of the immune system are secreted proteins, such as antibodies, and many important functions of the immune system are dependent upon the action of secreted proteins. For example, Saxon et al., Biochem Soc Trans 1997 May;25(2):383-7, discusses secreted IgE proteins.
For a further review of secreted proteins, see Nilsen-Hamilton et al., Cell Biol Int Rep 1982 Sep;6(9):815-36.
Nodal Proteins
Nodal and Nodal-related proteins have functions in mesoderm and endoderm induction and formation, as well as subsequent organization of axial structures such as heart and stomach in early embryogenesis. It has been demonstrated that dorsal tissue in a developing vertebrate embryo contributes predominantly to the axial structures of the notochord and pre-chordal plate while it recruits surrounding cells to form non-axial embryonic structures. Recent genetic and molecular studies in mouse, zebrafish and Xenopus reveal the molecular evidences of Nodal signaling (Zhou et al, Nature 361: 543-547 (1993); Feldman et al, Nature 395: 181-185, (1998); Rebagliati et al, Dev. Biol. 199: 261-272 (1998); Rebagliati MR, et al., Proc. Natl. Acad. Sci. USA. 95: 9932-9937 (1998); Sampath et al, Nature 395: 185-189 (1998); Ezal et al, J. Biol. Chem. 275: 14124-14131 (2000); Takahashi et al, Development 127: 5319-5329 (2000)). When squint and cyclops of zebrafish Nodal-related genes become simultaneously disrupted, embryos fail to form mesoderm and endoderm. Nodal-related genes Xnr5 and Xnr6 have been reported to initiate inductive events in Xenopus Nierwkoop center. Mouse Nodal gene has been identified through the insertional mutation. One mouse Nodal mutant (a retrovirally-induced recessive prenatal lethal mutation) fails to form mesoderm, and its embryonic ectoderm over-proliferates and subsequently degenerates.
The Nodal and Nodal-related proteins have been identified to use extracellular factors of transforming growth factors beta (TGF-beta) for signaling relay. Nodal signaling pathway is suggested as following (Stemple, Curr. Biol. 10: R843-846 (2000); Alexander and Stainier, Curr. Biol. 9:1147-1157 (1999) ). Nodal ligands interact with their co-factors to activate activin type I and type II or related receptors, which phosphorylate Smad2. The phosphorylated Smad2 forms a complex with Smad4 for nuclear import. In the nucleus, the Smad2/Smad4 complex serves as an effector of TGF-beta signaling by regulating transcription factors Fast-1 and Mixer, which control the expression of the genes for the development of dorsal axial structures and left-right asymmetry.
During gastrulation, the three germ layers of the embryo are formed and organized along the anterior-posterior body axis. In the mouse, gastrulation involves the delamination of ectodermal cells through the primitive streak and their differentiation into mesoderm. These processes do not occur in embryos homozygous for a retrovirally induced recessive prenatal lethal mutation, the strain 413-d insertional mutation. Instead of giving rise to mesoderm, embryonic ectoderm in 413-d mutants overproliferates and then rapidly degenerates, although extraembryonic lineages remain viable. For more information, see Dubois, et al., J Biol Chem. 1995 May 5;270(18):10618-24.
Nodal, a member of the transforming growth factor beta (TGF-beta) superfamily, is implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification. Experimental evidence has demonstrated that nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-beta. However, nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins. Furthermore, it has also been demonstrated that nodal induces p(CAGA)(12), a reporter previously shown to be specifically activated by Smad3. Expression of a dominant negative Smad2 significantly reduces the level of luciferase reporter activity induced by nodal treatment. Also, experimental evidence has shown that nodal signaling rapidly leads to the phosphorylation of Smad2. These results provide direct biochemical evidence that nodal signaling is mediated by both activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidence has shown that the extracellular cripto protein is required for nodal signaling, making it distinct from activin or TGF-beta signaling. For more information, see Sirotkin, et al., Curr Biol. 2000 Sep 7;10(17):10514; and Kumar A, et al., J Biol Chem. 2001 Jan 5;276(l):656-61.
Nodal-related signals comprise a subclass of the transforming growth factor-beta (TGF-beta) superfamily and regulate key events in vertebrate embryogenesis, including mesoderm formation, establishment of left-right asymmetry and neural patterning. Nodal ligands are thought to act with EGF-CFC protein co-factors to activate activin type I and II or related receptors, which phosphorylate Smad2 and trigger nuclear translocation of a Smad2/4 complex. The winged-helix transcription factor forkhead activin signal transducer-1 (Fast-1) acts as a co-factor for Smad2. Xenopus Fast-1 is thought to function as a transcriptional effector of Nodal signals during mesoderm formation, but no mutations in the Fast-1 gene have been identified. We report the identification of the zebrafish fast1 gene and show that it is disrupted in schmalspur (sur) mutants, which have defects in the development of dorsal midline cell types and establishment of left-right asymmetry. We find that prechordal plate and notochord are strongly reduced in maternal-zygotic sur mutants, whereas other mesendodermal structures are presentxe2x80x94a less severe phenotype than that caused by complete loss of Nodal signaling. These results show that fast1 is required for development of dorsal axial structures and left-right asymmetry, and suggest that Nodal signals act through Fast1-dependent and independent pathways. For more information, see Sirotkin, et al., Curr Biol. 2000 Sep 7;10(17):1051-4.
Secreted proteins, particularly members of the nodal-related secreted protein subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of secreted proteins. The present invention advances the state of the art by providing previously unidentified human secreted proteins that have homology to members of the nodal-related secreted protein subfamily.
The present invention is based in part on the identification of amino acid sequences of human secreted peptides and proteins that are related to the nodal-related secreted protein subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate secreted protein activity in cells and tissues that express the secreted protein. Experimental data as provided in FIG. 1 indicates expression in hypothalamus, teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled fetal lung, testis, and B-cell.